Quantitating Volatile Phenols in Cabernet Franc Berries and Wine

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Quantitating Volatile Phenols in Cabernet Franc Berries and Wine after On-Vine Exposure to Smoke from a Simulated Forest Fire Matthew Noestheden, Eric G Dennis, and Wesley Zandberg J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04946 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

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Journal of Agricultural and Food Chemistry

Quantitating Volatile Phenols in Cabernet Franc Berries and Wine after On-Vine Exposure to Smoke from a Simulated Forest Fire Matthew Noestheden1, 2, Eric G. Dennis1 and Wesley F. Zandberg1* 1

University of British Columbia Okanagan, British Columbia, Canada

2

Supra Research & Development, British Columbia, Canada

* Corresponding author contact details: Wesley Zandberg 1177 Research Rd Chemistry Department University of British Columbia Okanagan Kelowna, BC V1V 1V7 Canada [email protected] 250-807-9821 (t)

1 ACS Paragon Plus Environment


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Abstract

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Smoke-taint is a wine defect linked to organoleptic volatile phenols (VPs) in Vitis vinifera L.

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berries that have been exposed to smoke from wildland fires. Herein, the levels of smoke-taint-

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associated VPs are reported in Cabernet Franc berries from veraison to commercial maturity and

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in wine after primary fermentation following on-vine exposure to simulated wildland fire smoke.

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VPs increased after smoke exposure, were rapidly stored as acid-labile conjugates, and the levels

7

of both free VPs and conjugated forms remained constant through ripening to commercial

8

maturity. An increase in total VPs after primary fermentation suggested the existence of VP-

9

conjugates other than the acid-labile VP-glycosides already reported.

This conclusion was

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supported with base hydrolysis on the same samples. Relative to published results, the data

11

suggested a multifactorial regional identity for smoke-taint and they inform efforts to produce a

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predictive model for perceptible smoke-taint in wine based on the chemical composition of

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smoke-exposed berries.

14 15

Keywords

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Vitis vinifera, smoke, volatile phenol, wine, smoke taint, glycoside

17 18


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Introduction

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Volatile phenols (VPs) are organoleptic compounds that are found in Vitis vinifera L. (V.

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vinifera) berries and wine, originating from both endogenous and/or exogenous sources.

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Endogenous VPs are plant secondary metabolites involved in host stress response and

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reproduction1,2, whereas exogenous VPs can originate from: 1) the barrel aging of wine3, where

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the increased VP concentrations impart desirable sensory attributes; 2) biosynthesis during

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fermentation4 by Brettanomyces bruxellensis, which results in a wine defect known colloquially

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as ‘Brett-taint’5; or 3) the exposure of V. vinifera berries to smoke from wildland fires or

27

prescribed burns. When smoke-exposed berries are fermented, the resulting wine may possess

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negative sensory attributes (e.g., ‘smoky’, ashy’, ‘burnt meat’ and ‘Band-Aid’ aromas) or a

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paucity of varietal characteristics that, collectively, comprise a wine defect known as smoke

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taint.6–9 The impact of smoke-exposure to the wine industry is (and will remain) significant, as

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smoke-taint has been reported in North and South America, Australia and South Africa, all of

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which are important wine growing regions where the frequency of wildland fires are projected to

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increase.10

34

The exogenous VPs observed in smoke-exposed berries and/or the wines produced from

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these berries have been hypothesized to originate from the thermal degradation of lignin during

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wildland fires. The three monolignol subunits (sinapyl, p-coumaryl and coniferyl alcohols) can

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decompose to yield a variety of syringyl, alkylhydroxyphenyl and guaiacyl VPs (Figure 1).

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Since the ratio of monolignols changes as a function of plant species, the combustion of different

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fuel sources will expose berries to different VP ratios. For example, Kelly et al. showed that the

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combustion of Pinus radiata yielded different VP ratios in smoke than Eucalyptus species.11

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This suggests that smoke-taint may possess a regional identity based in part on the flora available



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for combustion during a wildland fire.

Such regionality could impact the performance of

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algorithms used to predict the potential for smoke-exposed berries to produce smoke-tainted

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wine. As most of the published data on smoke-taint comes from Australia, where wildland fires

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generally consume grasses and Eucalyptus trees, an evaluation of fuel sources relevant to other

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wine growing regions is required. Additionally, while Kelly et al. looked at different fuel

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sources, they did not evaluate the composition of the smoke-exposed berries prior to

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fermentation.11 This leaves a critical knowledge gap in the movement of VPs from smoke into

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berries and wine, especially since Kelly et al. observed VPs in wine that were not detected in

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their experimentally produced smoke.

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In planta and in wine, VPs are present in their free, volatile forms, but more dominantly they

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have been reported as an array of glycoconjugates.12 Kennison et al. analyzed VP concentrations

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in wine made from berries that were exposed to smoke at various times following the onset of

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veraison13 and at different phenological stages.14 However, they only looked at two or three VPs

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and did not look at the VP concentrations in berries prior to fermentation in either study. Indeed,

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with one exception6, most studies focus on the concentration of VPs in wine rather than in

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berries, since evaluating wine permits the direct correlation of VP concentrations with specific

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sensory characteristics.13–15 Alternatively, Dungey et al. looked at the change in guaiacyl-

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glycosides from the time of smoke-exposure until harvest, but did not evaluate other VP-

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glycosides.16 Since free VPs can be liberated during fermentation, with subsequent impact to the

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sensory characteristics of the resulting wine8, the ‘sensory potential’ of glycosidically-bound VPs

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is a critical component of smoke-taint. Accounting for all VP-glycosides is, therefore, necessary

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when assessing the potential for smoke-exposed berries to produce smoke-tainted wine.


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Our research group recently published a validated analytical method to accurately quantitate

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free VPs and the VPs released following acid hydrolysis (e.g., VP-glycosides), with the goal of

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developing a model to predict the probability of producing smoke-tainted wines from smoke-

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exposed berries based on the chemical composition of the berries prior to fermentation.17 In the

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present study, we demonstrate the application of these methods to the analysis of smoke-exposed

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Cabernet Franc berries and matched controls from the same vineyard, where the smoke was

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generated from a regionally-relevant fuel source. We report the levels of free and bound VPs (as

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determined by acid hydrolysis) at multiple time points, including immediately preceding and

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post-smoke exposure, through to commercial maturity and to the end of primary fermentation.

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To the author’s knowledge this is the first study to address such temporal data with demonstrably

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accurate methods for assessing a wide range of VPs (free and acid-labile). We hypothesize that

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the detailed elucidation of the time-dependent VP metabolic flux will aid in the development of

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predictive risk-assessment models in the event of smoke exposure.

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Materials and Methods

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Chemical and General Details. The following chemicals were purchased from Sigma

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Aldrich (Saint Louis, MO, USA) and used as received: HPLC-grade methanol (MeOH),

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isopropanol (IPA), acetonitrile (ACN), carbon disulfide, hexane, ethyl acetate (EtOAc),

81

hydrochloric acid, 2-methoxyphenol (guaiacol), 2,6-dimethoxyphenol (syringol), 2-methoxy-4-

82

methylphenol (4-methylguaiacol), 2-methoxy-4-ethylphenol (4-ethylguaiacol), 4-ethylphenol, 2-

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methoxy-4-allylguaiaicol (eugenol), 4-methylphenol (p-cresol) and 2-methylphenol (o-cresol).

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The internal standards d7-o-cresol and d7-p-cresol were purchased from Toronto Research

85

Chemicals (Toronto, ON, Canada). Details for the synthesis of d3-syringol and the model

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glycosides (guaiacyl-β-O-D-glucopyranoside, d3-guaiacol-β-O-D-glucopyranoside, p-cresyl-β-O5 ACS Paragon Plus Environment


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D-glucopyranoside,

syringyl-β-O-D-glucopyranoside, syringyl-gentiobioside, 4-ethylphenyl-β-O-

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D-glucopyranoside,

eugenyl-β-O-D-glucopyranoside) are reported elsewhere.17 Synthetic details

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for d3-guaiacol-β-D-glucopyranoside are summarized in the Supporting Information. Unless

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otherwise noted water was provided by a Barnstead E-Pure water purification system (Thermo

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Fisher Scientific; Waltham, MA, USA).

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ME204E analytical balance (Thermo Fisher Scientific). A Mettler Toledo FE20 FiveEasy pH

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meter was used to measure pH. An Allegra X-12R centrifuge was used for sample preparation

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(Beckman Coulter, Mississauga, ON, Canada).

Weighing was performed using a Mettler Toledo

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Stock and Calibration Solutions. Concentrated VP and isotopic internal standard (ISTD)

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stock solutions were prepared in IPA (d3-syringol in MeOH) at 1.0 – 10.0 mg/mL. An ISTD

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intermediate stock solution was prepared at 5 µg/mL in IPA. This solution was added to all

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calibration samples and extracts with a 100-fold (v/v) dilution. A 100 µg/mL intermediate

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calibration stock solution for GC-MS/MS was prepared in IPA. Calibration samples from 1 –

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1000 ng/mL were prepared fresh daily by diluting the intermediate stock solution into 1:1

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hexane:ethyl acetate. ISTD was added to each vial and the calibration samples were analyzed

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without further work-up. Model glycoside stock solutions were prepared in IPA at 1 mg/mL.

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All stock solutions were stored at -20 °C.

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Study Design and Smoke Exposure. Ten Cabernet Franc vines (clone 214, rootstock 204,

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planted in 2005, located near 49.1233° N, 119.5509° W) were split into five control plants and

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five plants that were exposed to experimentally produced smoke (vide infra), with the two

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groups separated by one panel of vines. Two bunches of berries were collected per vine for five

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time points from immediately preceding smoke application (~7 days post-veraison) until

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commercial maturity (23.5 ± 1.15 °Bx; Table S1). Samples were stored in separate polyethylene


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bags on ice prior to processing. In each test group (control and smoke-exposed) only four of five

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Cabernet Franc vines yielded sufficient berries to permit vinification after completing the

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analysis of berries collected at each time-point following smoke exposure.

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Fuel for the generation of smoke was collected from Pinus ponderosa (P. ponderosa) forests

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located near 49.9398° N, 119.3968° W and 49.8183° N, 119.4373° W. An amalgamated P.

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ponderosa fuel source was prepared by combining soil organic matter (SOM; 50% w/w), needles

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(20% w/w) and bark (30% w/w; cut into 3 cm pieces). This fuel mixture was stored in a

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polyethylene bag under ambient conditions.

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Vines were exposed to smoke for 60 min using a purpose-built, modular enclosure comprised

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of PVC/steel structural elements and a translucent polyethylene cover (Figure S1).

The

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enclosure was 18.3 m3 (1.1 m x 1.8 m x 9.1 m [w x h x l]). Smoke from our amalgamated fuel

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source was produced in a Masterbuilt Pro commercial food smoker (Masterbuilt Manufacturing;

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Columbus, GA, USA) and directed from the smoker exhaust vent into the modular enclosure via

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flexible aluminum ducting containing an inline fan (0.003 – 0.005 m3/s); the fan speed was

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adjusted as necessary to maintain consistent smoke density within the enclosure. Smoke density

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(PM2.5), relative humidity and temperature in the enclosure were monitored using an AirBeam

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wearable air monitor (HabitatMap; NY, USA). Smoke was sampled in triplicate using Orbo 33

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activated petroleum carbon (Sigma Aldrich, 700 mg/390 mg, 8 x 150 mm), with the identical

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primary and secondary sorbent beds separated by a foam insert and each end of the sorbent beds

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contained with glass wool. Sampling was done at 250 mL/min for 60 minutes using a Gilian

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GilAir-3 sampling pump (Sensidyne, LP; FL, USA). Sorbent tubes were stored at 4 °C until

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analysis. The temperature inside the smoke enclosure did not exceed 30 °C and the relative

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humidity stayed above 25% (Figure S2). Smoking was done early in the day (~ 6:00 am),



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approximately seven days after the onset of veraison, which was determined by the vineyard.

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Control vines were not enclosed in the smoking structure (without smoke) due to a strong

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residual smoke aroma in the polyethylene cover after use.

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Sample Preparation and Analysis.

Samples were analyzed for free VPs using gas

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chromatography tandem-mass spectrometry (GC-MS/MS) and for intact VP-glycosides using

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ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry

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(uHPLC-QToF). Method details are summarized in the Supporting Information (Tables S2 –

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S5).

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On the same day as sample collection, berries were stemmed, a subset of 100 berries was

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weighed and the degrees brix (° Bx, n = 4 berries/vine) were recorded using an Atago PAL-1

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refractometer (Atago USA, Inc.; Bellevue, Washington, USA). Also on the same day as sample

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collection, berries were prepared as whole berry homogenate (HMG) by homogenizing whole

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berries in a Black & Decker BL2010BGC commercial blender (Stanley, Black & Decker; New

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Britain, CN, USA) for 60 s. Prior to preparation as HMG, samples from t0 – t2 (Table S1) were

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divided into subsets, with one set untreated and the other washed with gentle agitation in tap

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water for 30 s. All samples were stored at -20 °C in 50 mL polypropylene tubes (Sarstedt;

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Nümbrecht, Germany) after preparation for up to four months prior to analysis. Berries intended

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for vinification were stemmed and stored at -20 °C in polypropylene bags for up to 12 months.

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The primary and secondary Orbo 33 sorbent beds were collected into separate 4 mL glass

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vials. Each vial received 2 mL of carbon disulfide and was incubated under ambient conditions

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for 60 min with periodic agitation after the addition of 50 ng/g VP ISTD. The resulting extract

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was analyzed for VPs using targeted GC-MS/MS.


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HMG extracts of free and acid-labile VPs for GC-MS/MS were prepared according to

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Noestheden, et al..17 Wine extracts were prepared the same way, except the extract was prepared

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in a 1:1 ratio (sample:extraction solvent) rather than a 5:2 ratio. The bound fraction of VPs was

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calculated from the difference between the free and total VPs (i.e., the VPs measured after acid

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hydrolysis) for all samples.

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Base hydrolysis was performed on HMG, wine and 1 µg/mL model glucosides by adding 1

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mL of 1 M NaOH to 5 mL of sample. After 4 hours at 100 °C the base hydrolyzed samples were

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neutralized with 2 mL of 1 M NaH2PO4. Samples intended for GC-MS/MS were fortified with

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50 ng/g VP ISTD and extracted. Samples for LC-MS/MS were fortified with 300 ng/mL d3-

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guaiacol-β-D-glucopyranoside and analyzed without further work-up.

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Yeast. Yeast cultures were prepared by streaking-plating yeast (strain EC1118) onto solid

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YEPD media (10% yeast extract, 10% bacterial peptone, 20% dextrose, 20% agar), followed by

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incubation at 28 °C for 48 hours, then storage at 4 °C. A subsample of the yeast culture was

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collected with a sterile spatula and suspended in sterile water. The yeast culture was then

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adjusted to 2.0 absorbance units at 600 nm by dilution with sterile water.

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Microvinification. Micro-scale wines for the control and smoke-exposed CF berries (n = 1)

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were prepared as follows. Erlenmeyer flasks (500 mL) were sanitized by exposure to 20 mL of

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5% sodium metabisulfite solution for one hour and rinsed thoroughly with water. For each of the

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berry samples from control and smoke-exposed vines, 100 g aliquots of frozen berries were

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weighed into a sanitized Erlenmeyer flask and 5 mL of a 1% sodium metabisulfite solution was

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added. The berries were thawed for 24 h at 4 °C, crushed with a spatula and, where necessary,

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the pH of the juice was adjusted to 3.6-3.8 by the addition of 10% aqueous tartaric acid (< 500

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µL). The resulting musts were inoculated with yeast culture (1 mL, adjusted to 2.0 AU at 600



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nm). Flasks were then fitted with air locks and fermentations were allowed to proceed with

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twice daily agitation until mass loss stabilized (ca. 14 d). Fermentation was halted by removing

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yeast cells by centrifugation (3000 x g for 15 min). The clarified wines were then stored in glass

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at 4 °C under argon prior to analysis.

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Method Performance. Calibration curves were fit using linear or quadratic regression and

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inverse concentration weighting. The limit of detection (LOD) and limit of quantitation (LOQ)

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calculations were adapted from Evard et al.. as18:

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‫ܦܱܮ‬௜ /‫ܱܳܮ‬௜ = ݇ × ‫ݏ‬௡

(1)

186

Where ‫ݏ‬௡ was the standard deviation of n spike-recovery extracts for compound i and k was a

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multiplier equal to 3.3 for the LOD and 10 for the LOQ. Accuracy and precision for the analysis

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of VPs in wine were evaluated by fortifying model wine (MW; for contents see Table S6) at 5,

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20 and 200 ng/g (n = 5/concentration). Recoveries were corrected for a MW density of 0.98

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g/mL (empirically determined).

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Data Acquisition and Processing. GC-MS/MS data was processed using the Xcalibur

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(version 3.0.63) and TraceFinder (version 3.2.512.0) software packages (Thermo Scientific).

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The uHPLC-QToF data acquisition and processing were carried out using the MassHunter

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Workstation software suite (Agilent Technologies), with version numbers as follows: Data

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Acquisition Workstation (v B.08.00) and Qualitative Analysis (v B.07.00, Service Pack 2). Data

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reduction and statistical calculations were performed using Microsoft Excel (Microsoft

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Corporation; Redmond, WA, USA) and BoxPlotR19. Where relevant, data are displayed ± 1

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standard error of the mean (SEM). Unless noted, all statistical comparisons were done using a

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Mann-Whitney U test (α = 0.05).


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Results and Discussion

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Simulating Wildland Fire Smoke. There are three types of wildland fires: 1) ground fire,

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where soil organic matter (SOM) is consumed; 2) surface fire, where short vegetation and SOM

203

are consumed; and 3) crown fire, where tall trees and shrubs are consumed largely independently

204

from SOM.20,21 All three types may co-exist during a given wildland fire and the occurrence of

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one over another is a complex interaction between weather patterns, moisture, ignition source,

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type of forest, proximity to urban centers, etc.22 Given this complexity, the simulation of

207

wildland fire smoke in the present study was achieved using an amalgamated fuel containing P.

208

ponderosa bark (30% w/w), needles (20% w/w) and SOM (50% w/w). This approach deviated

209

from the widely referenced study by Kennison et al., who selected barley straw to produce

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smoke with a consistent composition, relative to that produced during the combustion of native

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vegetation.9 As the lignin composition of barley straw (17%)23,24 is less than wood (23 – 45%),11

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and as the ratio of monolignol subunits in barley and wood differ, the VPs produced from each

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fuel source under a given set of combustion parameters are likely to change.11 Given the

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prevalence and proximity of ponderosa pine forests to vineyards in the western United States and

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Canadian wine growing regions, it was hypothesized that our amalgamated P. ponderosa fuel

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source would be more relevant to North America. Sheppard et al.25 used P. ponderosa to study

217

smoke-taint, but they used a chipped and homogenized fuel containing all parts of the tree and

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omitted SOM. In summary, an amalgamated fuel will provide a better approximation of the

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variability in VPs produced during a given wildland fire event, although, it should be noted that

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none of the approaches used to date (including the one used herein), fully account for

221

environmental influences or the variability in the lignin composition of SOM26–29 as they relate

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to the generation of VPs in smoke.



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Targeted GC-MS/MS analysis of the smoke generated from our P. ponderosa fuel source

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showed the presence of o/p-cresol, guaiacol, 4-methylguaiacol and 4-ethylguaiacol (Figure 2).

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While the VPs identified were consistent with the smoke generated upon combustion of Pinus

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radiata11, the absence of several VPs and differences in the ratios of detected VPs supported the

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presence of regional differences in the presentation of smoke-taint. Further supporting this idea

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was the lack of 4-methylsyringol, which was identified as major sources of total VPs in the

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smoke, berries and wine of smoke-exposed Chardonnay and Cabernet Sauvignon.30 No evidence

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of 4-methylysringol was found in the smoked-exposed CF berries used in this study, or in the

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experimental smoke (data not shown). It is not clear if these differences were a function of

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environmental, varietal, or experimental parameters (e.g., fuel source).

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The reproducible application of smoke to V. vinifera berries on-vine is a logistical challenge,

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as controlling the volatile organic chemical composition (i.e., VPs) of smoke is

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multifactorial.31,32 Nephelometry has previously been used as an aggregate measure of smoke

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exposure, with Kennison et al. demonstrating that a single 30 min exposure at 200 µg/m3

237

(particulate matter ≤ 10 µm, PM10) was sufficient to yield 90 µg/L guaiacol in wine made from

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the smoke-exposed berries.13 In the present study, PM2.5 values were measured during smoke

239

application, but high variance and frequent detector saturation limited the usefulness of the

240

results (Figure S2). Given these results and the current lack of correlative studies between

241

PM2.5/10 values and the magnitude of VPs in smoke-exposed berries, it was decided that smoke

242

application would best be reported as the amount of fuel consumed (500 g) over a set time (60

243

min) inside a given volume (18.3 m3).

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Cabernet Franc Field Trials. Cabernet Franc (CF) vines were exposed to simulated forest

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fire smoke approximately seven days after the onset of veraison, as this is when the berries are


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most susceptible to increases in total VPs with smoke exposure.13 To the best of our knowledge,

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this is the first report on the impact of smoke-exposure to CF berries and wine. Anecdotal

248

evidence from growers in the Okanagan Valley of British Columbia, Canada suggested that

249

smoked-exposed CF berries were more likely to yield smoke-tainted wine when compared with

250

other varietals exposed to similar smoke. For all samples, an average weight from 100 berries

251

was recorded, as were the °Bx. When compared against the VP data no trends were observed

252

between these metrics and the concentration of free or bound VPs, nor did the smoke-exposed

253

berries differ significantly in °Bx or average berry mass from control berries (data not shown).

254

These results were consistent with a study that evaluated the impact of smoke-exposure on vine

255

physiology.6

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Washed versus Unwashed Berries. It has been demonstrated that VPs can be translocated

257

into berries and leaves when exposed directly to solutions of guaiacol.33 Despite this evidence of

258

direct uptake, it remains unclear if particulates from wildland fires act as vehicles to transport the

259

VPs to the berry, or if the VPs are in their free form leading to direct uptake by the berry. Using

260

smoke-exposed berries that were harvested immediately after smoke application, the effect of

261

washing the berries was investigated as: 1) a model to test if overhead irrigation following smoke

262

exposure could help mitigate the severity of smoke-taint; and 2) an indirect test of the role played

263

by particulates (Figure S3). For VPs above their respective LOQs, the washed and unwashed

264

berries showed the same concentrations of VPs. Evaluation of berries immediately after on-vine

265

smoke-exposure has yet to be reported, so the kinetics of VP uptake have only been observed on

266

the scale of days and weeks6,13,14,25 rather than hours.

267

suggested that regardless of how they arrive at the berry (particle-bound or free), VPs strongly

While not definitive, these results



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adhere to and/or rapidly translocate into the berry. This indicated that overhead irrigation after a

269

smoke-exposure event is unlikely to decrease the VPs that are concentrated in the berry.

270

Free versus Bound VPs. Several studies have examined how VPs are stored and/or are

271

biochemically modified (e.g., glycosylated) in smoke-exposed berries and wine at different time-

272

points between veraison and commercial maturity. Those studies looking at free VPs were from

273

the infancy of smoke-taint research and only examined guaiacol, 4-methylguaiacol and 4-

274

ethylphenol.13,14,25 Subsequent publications highlighted strong correlations between the intensity

275

of typical smoke-taint characteristics in wine and the presence of syringol and cresols6,34,35,

276

making their absence from these earlier studies noteworthy. Furthermore, with their focus on the

277

impact of smoke at key developmental stages, these initial studies lack data on the evolution of

278

VP storage forms during ripening and fermentation. Dungey et al. looked at the same vines over

279

time following smoke-exposure, but only evaluated guaiacyl-glycosides.16

280

evaluation of the time-dependent biochemical dynamics of VPs within the same smoke-exposed

281

vines remains unexamined. This information is critical to the development of a robust smoke-

282

taint risk-assessment model. Therefore, to fill this gap, the present study applied our recently

283

developed analytical methods17 to quantitate a broad range of free and bound VPs (as determined

284

by acid hydrolysis) in the same control and smoke-exposed CF vines over a time span

285

encompassing nearly all of the post-veraison ripening process until harvest and to the end of

286

primary fermentation (Figure 3; see Table S7 for a numerical summary).

To date, an

287

Examining these data broadly, it was intriguing that the ratio of VPs detected in the

288

experimental smoke (Figure 2) were not reflected in the ratio of free, bound or total VPs detected

289

in smoke-exposed berries. This was most apparent for syringol, which was not detected in the

290

smoke reported here, nor was it found in the work of Kelly et al.11, despite appearing at high


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concentrations in the wine produced for both studies. Moreover, analysis of VPs in berries and

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wines revealed that the relative VP ratios changed following primary fermentation.

293

comparing smoke-exposed berries with their development-matched controls, these results

294

suggested differential uptake and/or metabolism of smoke-borne VPs. We observed that in the

295

smoked-exposed berries there was a statistically significant increase in both free and bound

296

guaiacol over control samples, with the total guaiacol levels remaining constant until commercial

297

maturity (42 days after smoke exposure). Similar trends were observed for the cresols, although

298

the free form of o-cresol was present below its’ LOQ in all except the 7-day sample. 4-

299

Methylguaiacol also displayed similar results for the free VP. Conversely, the bound form of 4-

300

methylguaiacol trended upwards until commercial maturity rather than holding constant, with

301

statistical significance when comparing the 4-methylguaiacol levels at commercial maturity

302

against those measured immediately post-smoke exposure (i.e. 0.04 days). With the exception of

303

4-ethylphenol, the levels of acid-labile VPs in berries exceed those of their free analogues. The

304

in-berry levels of three other VPs, 4-ethylguaiacol, eugenol, and syringol did not permit the

305

discrimination between smoke-exposed and control berries, with 4-ethylguaiacol being detected

306

in only two of ten samples and eugenol/syringol displaying equal levels between all samples and

307

time points.

Upon

308

The guaiacol and cresol data demonstrated that the ratio of bound-to-free VPs was

309

established during smoke exposure and remained constant until commercial maturity. Assuming

310

the acid-labile compounds (i.e., bound guaiacol) were simple glycosides, these results diverged

311

from those previously reported for guaiacyl-glycosides, which were shown to increase in

312

concentration for at least two weeks after smoke exposure in Merlot and Voignier berries.16 In

313

contrast to the trends observed for guaiacol and o/p-cresol, the bound-to-free VP ratio for 4-



Page 16 of 33

314

methylguaiacol increased following smoke exposure. Although this increase in the bound form

315

of the VP was consistent with published guaiacyl-glycoside results16,

316

corresponding decrease in the levels of free VP indicated that at least some of the acid-labile 4-

317

methylguaiacol conjugates observed in the berries at commercial maturity could be derived from

318

an alternate pool of VP conjugates that was resistant to acid hydrolysis.

the absence of a

319

In addition to evaluating the free and bound VPs in CF berries, the levels of free and bound

320

(acid-labile) VPs in wines (primary ferments) made from the control and smoke-exposed berries

321

harvested at commercial maturity were also determined (Figure 3). This necessitated adaptation

322

and validation of the GC-MS/MS methods developed for berries by Noestheden et al.17 to

323

include the analysis of VPs in wine (Table S9). Our analysis revealed that, with the exception of

324

4-ethylphenol, the levels of free VPs in wines made from smoke-exposed berries were

325

statistically higher than the free VPs measured in the same berries prior to fermentation. The

326

increase in free VP concentrations ranged from 4-fold for o-cresol to 150-fold for syringol.

327

Surprisingly, in the case of guaiacol, syringol and eugenol the levels of the free VPs in wines

328

exceeded those of total VPs in the berries, an observation that intriguingly held for both the

329

control and smoke-exposed berries. With the exception of eugenol, the levels of free VPs

330

detected in wines produced from smoke-exposed berries were statistically higher than the

331

corresponding control wines, with increases in free VP concentrations ranging from 2.4-fold for

332

guaiacol to 8.5-fold for p-cresol. While the present study did not focus on sensory analysis, the

333

free guaiacol levels measured in wine made using the smoke-exposed CF berries relative to the

334

reported aroma threshold of guaiacol in red wine suggested that a quantifiable sensory impact

335

would be observed.13


Page 17 of 33


336

Previous smoke-taint research suggested that free VPs detected in wine may be produced

337

from glycosidically-bound precursors by the action of microbial glycosidases8 and/or acid

338

hydrolysis during fermentation and aging.15,36 Therefore, it was hypothesized that the increase in

339

free VPs observed in our CF wines (relative to berries) should be accompanied by a

340

corresponding decrease in the levels of bound (acid-labile) VPs observed after fermentation.

341

However, such a decrease was not observed in the smoke-exposed CF wines. For example, the

342

in-wine levels of acid-labile forms of guaiacol, and o/p-cresol all significantly exceeded those

343

observed in berries, while the levels of bound 4-methylguaiacol and eugenol remained

344

effectively unchanged. As observed for the free VPs, in several instances (guaiacol, o/p-cresol,

345

syringol, 4-ethylguaiacol and eugenol) acid-labile VPs were also detected in control wines even

346

though they were present below or just above their LOQs, in the same berries prior to

347

fermentation. It is worth noting that 4-ethylguaiacol (Figure 3) showed a trend (albeit not

348

statistically significant) in wine towards higher concentrations in the smoke-exposed berries,

349

despite none of this VP being observed above the LOQ in either control or smoke-exposed

350

berries. Note, however, that 4-ethylguaiacol was in the P. ponderosa fuel source (Figure 2).

351

Noestheden et al. previously demonstrated the quantitative recovery of free VPs following

352

acid hydrolysis of VP-glycosides.17 Therefore, the increase in total VPs (inclusive of acid-labile

353

conjugates and free forms) following primary fermentation suggested the presence of VP storage

354

forms other than simple acid-labile glycosides. In the present study, the data indicated that these

355

alternate storage forms were more relevant to the final VP concentration in wines than VP-

356

glycosides, given the large increase in bound VPs (up to 16-fold for guaiacol) in wine made from

357

smoke-exposed CF berries. The increase in acid-labile VP-conjugates after primary fermentation

358

suggested that microbial enzyme-catalyzed transformations not only increased the levels of free



Page 18 of 33

359

VPs (as previously demonstrated8,34,37), but that they also produce a larger pool of acid-labile

360

VP-conjugates. It is plausible that this alternate VP storage form could also explain the increase

361

in acid-labile 4-methylguaiacol conjugates observed in smoke-exposed berries during the

362

ripening process (Figure 3), with differential metabolic flux between the two VP storage forms

363

controlling the relative amounts of each observed during the ripening process.

364

Base Hydrolysis.

As confirmation that the alternate VP-conjugates were not simple

365

glycosides, the CF berries (control and smoke-exposed) and their corresponding wines were

366

subjected to base hydrolysis (pH 11.5, 4 h, 100 °C). Prior to initiating this experiment, the

367

stability of the free VPs and a set of model VP-glycosides under base hydrolytic conditions were

368

evaluated (Table 1). This experiment demonstrated that free VPs were stable to the basic

369

hydrolysis conditions used and could be quantitatively recovered prior to GC-MS/MS analysis.

370

The stability of the model glycosides under basic conditions depended on the nature of the VP

371

aglycone. 4-Ethylphenyl, eugenyl and p-cresyl glucosides were quantitatively recovered from

372

basic digestion conditions, whereas the guaiacyl-glucoside and syringyl-glycosides displayed

373

varying levels of base sensitivity, respectively exhibiting 30% and 70% loss of the glycosides as

374

assessed by uHPLC-Q-ToF. However, in these instances the loss of the glycosides did not

375

correspond to a quantitative increase in free VPs, with only 39% of the lost guaiacyl-β-D-

376

glucopyranoside, 5% of the syringol-β-D-glucopyranoside and 55% of the syringol gentiobioside

377

recovered as free VP (data not shown). This result was consistent with observations that the

378

base-catalyzed hydrolysis of glycosidic bonds can require more aggressive conditions (170 °C,

379

2.5 M NaOH, > 101 kPa38) than employed in this study, depending on the nature of the glycone

380

and aglycone.39,40 The alkaline hydrolytic behavior of the VP-glycosides reported herein should

381

be applicable to other matrices, as all reactions were performed at the same pH, regardless of


Page 19 of 33


382

matrix (e.g., berries, wine or model wine). It follows from these observations that, with an

383

accommodation for the observed glycoside degradation and its subsequent impact on the

384

recoveries of guaiacol and syringol in base, an increase in free VPs under these alkaline

385

conditions would confirm the presence of a VP-conjugate other than a simple glycoside.

386

The base hydrolysis data for free guaiacol demonstrated that there were base-labile

387

conjugates in berries and wine (Figure 4).

For example, the statistically equivalent

388

concentrations of guaiacol released after the acid hydrolysis of wine made using smoke-exposed

389

berries (152 ± 13.7 ng/g) and the base hydrolysis of those berries (124± 10.9 ng/g) suggested that

390

the base-labile pool of bound VPs was modified during fermentation to make them acid-labile.

391

Similar results were obtained for syringol, although in this instance the syringol concentrations

392

increased markedly (Figure 4; the syringol base hydrolysis data was scaled down 10-fold for

393

readability). Again, in keeping with the trends observed for guaiacol, the syringol data showed

394

the presence of a storage form that was not acid labile (i.e., not a simple glycoside) prior to

395

fermentation. It is plausible that the mechanism responsible for these observations can also be

396

used to explain why Kelly et al.11 observed high levels of syringol in wine despite observing low

397

syringol concentrations in smoke generated from Pinus radiata. The cresol data continued to

398

provide evidence of base-labile VP-conjugates that were made accessible to acid hydrolysis

399

during primary fermentation. In contrast, the 4-methylguaiacol results were inconclusive, as the

400

base labile data was only marginally greater than the free VP concentrations.

401

hydrolysis data did not yield significant results for eugenol, 4-ethylguaiacol and 4-ethylphenol

402

(Figure 4).

403

ongoing in our research group.

The base

Characterizing the identity of these abundant base-labile VP-conjugates is the



Page 20 of 33

404

Having more accurate and timely access to information regarding the potential of smoke-

405

exposed berries to produce smoke-tainted wine will give growers and wineries time to plan

406

suitable fermentation adjustments and/or viticultural changes to mitigate the impact of smoke

407

exposure.

408

concentrations in berries and wine could be established immediately after smoke exposure.

409

However, the base hydrolysis and fermentation data demonstrated the need to better elucidate the

410

metabolic fate of VPs in smoke-exposed berries to yield more definitive predictive correlations

411

between the VPs stored in smoke-exposed berries and the probability of producing smoke-tainted

412

wines. Further influencing the generation of predictive correlations is the regionality of smoke-

413

taint, with our data showing clear differences in VP profiles from those previously reported.

414

Indeed, the results herein suggest that applying existing smoke-taint research, while an excellent

415

starting point, may not be entirely applicable to the presentation of smoke-taint in all wine

416

producing regions. Studies aimed at establishing a correlation between total VP concentrations

417

in berries and perceptible smoke-taint in wine are ongoing in our research group.

The results presented herein suggested that correlations between total VP

418 419

Acknowledgements. MN and WZ would like to thank Supra Research & Development for GC-

420

MS/MS access.

421

preparation and Sydney Morgan for her assistance with yeast preparation.

We thank Ben Tiet and Katelyen Thiessen for their assistance in sample

422 423

Funding Sources. MN acknowledges the MITACS Accelerate program, University Graduate

424

Fellowship and the Walter C. Sumner Memorial Fellowship for funding. WFZ acknowledges

425

the Natural Science and Engineering Research Council of Canada (Discovery Grant, 2016-

426

03929, and Engage Grant, 509805-17) the British Columbia Wine Grape Council (research


Page 21 of 33


427

grant) for funding and the Canada Foundation for Innovation: John Evans Leaders Fund (project

428

number 35246) and the British Columbia Knowledge Development Fund for supporting critical

429

infrastructure.

430 431

Supporting Information. Synthetic details, analytical instrument parameters, field trial design

432

and metrics, analytical results, wine validation data.



Page 22 of 33

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Vogt, T. Phenylpropanoid biosynthesis. Mol. Plant 2010, 3 (1), 2–20.

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Jiranek, V. Smoke taint compounds in wine: Nature, origin, measurement and amelioration of affected wines. Aust. J. Grape Wine Res. 2011, 17 (2), 2–4.

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Kelly, D.; Zerihun, A.; Singh, D. P.; Vitzthum Von Eckstaedt, C.; Gibberd, M.; Grice, K.; Downey, M. Exposure of grapes to smoke of vegetation with varying lignin composition and accretion of lignin derived putative smoke taint compounds in wine. Food Chem. 2012, 135 (2), 787–798.

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validation of a HPLC-MS/MS method for the quantitative analysis of smoke-derived phenolic glycosides in grapes and wine. J. Agric. Food Chem. 2013, 61 (1), 25–33. (13)

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Kennison, K. R.; Wilkinson, K. L.; Pollnitz, A. P.; Williams, H. G.; Gibberd, M. R. Effect of smoke application to field-grown Merlot grapevines at key phenological growth stages on wine sensory and chemical properties. Aust. J. Grape Wine Res. 2011, 17 (2), 5–12.

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Dungey, K. A.; Hayasaka, Y.; Wilkinson, K. L. Quantitative analysis of glycoconjugate precursors of guaiacol in smoke-affected grapes using liquid chromatography-tandem mass spectrometry based stable isotope dilution analysis. Food Chem. 2011, 126 (2), 801– 806.

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Sheppard, S. I.; Dhesi, M. K.; Eggers, N. J. Effect of pre- and postveraison smoke exposure on guaiacol and 4-methylguaiacol concentration in mature grapes. Am. J. Enol. Vitic. 2009, 60 (1), 98–103. 23 ACS Paragon Plus Environment


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Page 26 of 33

Figure 1. A sample of the VPs reported in V. vinifera berries and wine after on-vine exposure of berries to wildland fire smoke.

Figure 2. Relative levels of VPs identifieda in the smoke generated from the combustion of P. radiata11, P. ponderosa b bark and needles, and soil organic matter from a P. ponderosa forest, as well as smoke-exposed CF berries and wine c. a

Identified using targeted GC-MS/MS analysis.

b

Relative concentration based on raw instrument response as a fraction of the response for all

detected VPs. c

Ratios taken from total VPs quantified in smoke-exposed CF berries at commercial maturity

and in wine made from the same berries.

Figure 3.

a, b

c

Total VPs expressed as free (light gray) and bound (dark gray) forms in Cabernet

Franc berries from immediately preceding on-vine smoke exposure until commercial maturity and to the end of primary fermentation (W). a

Data are reported as the mean ± 1 SEM and were tabulated from up to five biological replicates

(see Table S8). b

With the exception of syringol, the levels of both free and bound VPs c differed significantly

(Mann-Whitney U test, α = 0.05) between smoke-exposed (+) and control (-) groups. c

As determined by acid hydrolysis.


Page 27 of 33


Figure 4.

a, b

Box-whisker plots c of free VPs (white), acid-labile VP-conjugates (light gray) and

base-labile VP-conjugates (dark gray) in Cabernet Franc berries at commercial maturity (B) and after primary fermentation (W). a

b

c

Data were tabulated from up to five biological replicates (see Figure S8). Statistical difference from respective control (-) samples (Mann-Whitney U test, α = 0.05). th

th

Center lines show medians, box limits indicate the 25 and 75 percentiles, whiskers extend to th

th

1.5 times the interquartile range from the 25 and 75 percentiles, points beyond these ranges were assigned as outliers. d

Results are scaled down 10-fold for clarity at lower concentrations.



Page 28 of 33

Table 1. Recovery of free VPs and model glucosides from acid and base digests.a, b free VPs (%)

model glycosides (%)

compound

acid 17

base

acid 17

4-ethylguaiacol

89 ± 1.9

81 ± 3.9

4-ethylphenol

97 ± 4.1

90 ± 1.8

-

4-methylguaiacol

82 ± 3.9

93 ± 2.9

eugenol

92 ± 3.7

guaiacol

100 ± 1.4

c

c

base c

118 ± 12.0 c

90 ± 1.7

-

102 ± 3.6

88 ± 2.8

-

69 ± 5.4

o-cresol

103 ± 4.4

89 ± 1.9

p-cresol

92 ± 4.2

syringol

71 ± 2.3

c

c

88 ± 2.4

-

96 ± 7.8

100 ± 1.9

-

33 ± 11.1/25 ± 15.6 d

a

Acid and base digests were performed at 100 °C for 4 h, at pH 1.5 and 11.5, respectively. Values are reported as mean ± 1 standard error of the mean (n = 3). c Glucosides for these VPs were not synthesized. d Syringol-β-D-glucopyranoside/syringol gentiobioside. b


Page 29 of 33


Figure 1

compound

R1

R2

R3

4-ethylguaiacol

OMe

Et

H

4-ethylphenol

H

Et

H

4-methylguaiacol

OMe

Me

H

guaiacol

OMe

H

H

eugenol

OMe

C3H5

H

o-cresol

H

H

Me

p-cresol

H

Me

H

syringol

OMe

H

OMe



Page 30 of 33

Figure 2


Page 31 of 33


Figure 3



Page 32 of 33

Figure 4


Page 33 of 33


TOC Graphic


Quantitating Volatile Phenols in Cabernet Franc Berries and Wine

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